US20220154239A1 - Peptide for complexing zinc ion, complex thereof and use therefor - Google Patents

Peptide for complexing zinc ion, complex thereof and use therefor Download PDF

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US20220154239A1
US20220154239A1 US17/586,774 US202217586774A US2022154239A1 US 20220154239 A1 US20220154239 A1 US 20220154239A1 US 202217586774 A US202217586774 A US 202217586774A US 2022154239 A1 US2022154239 A1 US 2022154239A1
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peptide
zinc ion
zinc
complexing
solution
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Qingzhu Zeng
Yingmin Zheng
Qingling Xu
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Guangzhou University
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Guangzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/14—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
    • A23J1/148—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds by treatment involving enzymes or microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/16—Vegetable proteins from soybean
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/20—Inorganic substances, e.g. oligoelements
    • A23K20/30—Oligoelements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A23L33/165—Complexes or chelates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22002—Papain (3.4.22.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24028—Bacillolysin (3.4.24.28)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • a sequence listing is submitted as an ASCII formatted text filed via EFS-Web, with a file name of “Sequence_listing.TXT”, a creation date of Jan. 27, 2022, and a size of 486 bytes.
  • the sequence listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.
  • the invention relates to the technical field of biochemical engineering, in particular to a peptide for complexing zinc ion, complex thereof and use therefor.
  • zinc deficiency In China, the phenomenon of zinc deficiency is commonly seen among the crowd, and it is especially serious for children. Up to 39.6% of the children have different degrees of zinc deficiency, and the zinc deficiency rate is as high as 64.6% especially for children aged 3 to 4. For those aged 0 to 18, zinc deficiency ranks the first in trace element deficiencies with a zinc deficiency rate of 50% or more. Promoting the transport and absorption of the trace element zinc in the human body, and improving the absorption and the utilization rate of zinc are important scientific issues that have always been concerned with in the fields of food nutrition or functional foods. In addition, in order to avoid the crisis of illness caused by zinc deficiency in farmed livestock, poultry and aquatic animals, and so on, it is usually necessary to add zinc supplements to the feed. At present, inorganic zinc such as zinc oxide, zinc sulfate or zinc gluconate etc. is usually used as a zinc supplement for human or animal bodies.
  • Organic zinc is closer to the functional form of zinc inside the organism, which can prevent the formation of insoluble substances inside the organism due to the supplement of inorganic zinc. Moreover, organic zinc has a higher biological efficiency than that of inorganic zinc, and only a trace amount of organic zinc is necessary to achieve the effect of zinc supplementation, which avoids the harm caused by excessive zinc inside the body due to poor absorption of inorganic zinc.
  • active peptides that have affinity with zinc ions (Zn 2+ ), peptides for complexing zinc, and their complexes can both promote the transport and the absorption of zinc.
  • Peptide-zinc complex belongs to the category of organic zinc, is a new type of trace element enhancer that promotes the body's zinc intake, and is more helpful to promote the transport and the absorption of zinc by the body's cells.
  • the purpose of the present invention is to provide a peptide for complexing zinc ion, the complex thereof and use therefor.
  • a first aspect of the present invention provides:
  • a peptide for complexing zinc ion having an amino acid composition and sequence of SEQ ID NO:1: Lys-Tyr-Lys-Arg-Gln-Arg-Trp (KYKRQRW).
  • the above-described peptide for complexing zinc ion is derived from soybean, peanut or chemical synthesis. Certainly, other alternative amino acid and protein synthesis methods in this field can be reasonably selected according to actual needs, to obtain the above-mentioned peptide for complexing zinc ion.
  • a second aspect of the present invention provides:
  • a method for preparing a peptide for complexing zinc ion comprises steps as follows:
  • step (2) taking the protein peptide solution obtained in step (1), and subjecting it to separating, screening, and purifying, to obtain a peptide component, wherein the enzyme used for the enzymolysis is one or more selected from the group consisting of ALcalase enzyme, papain and Bacillus subtilis neutral protease; and the peptide component contains the above-described peptide for complexing zinc ion.
  • the enzyme used for the enzymolysis is one or more selected from the group consisting of ALcalase enzyme, papain and Bacillus subtilis neutral protease; and the peptide component contains the above-described peptide for complexing zinc ion.
  • the molecular weight of the peptide component in step (2) is 1 to 5 K Da; the content of Peptide is 15.58% to 17.18%; and the complexing rate of the zinc ion is 48.5% to 52.16%.
  • the molecular weight of the peptide component, the content of Peptide, and the complexing rate of the zinc ion can be reasonably selected according to actual needs.
  • the way for the above-described separating, screening, and purifying in step (2) is one or more selected from the group consisting of ultrafiltration fractionation, gel chromatography and high performance liquid chromatography.
  • the temperature is 35° C. to 55° C.
  • the pH is 6.0 to 8.5.
  • pH for the ALcalase enzyme is 7.0 to 8.5
  • pH for the papain is 6.5 to 7.5
  • pH for the Bacillus subtilis neutral protease is 6.0 to 7.0.
  • a third aspect of the present invention provides:
  • the preparation method thereof is: taking the above-described peptide for complexing zinc ion or the above-described peptide component, adding a zinc ion solution, and adjusting the pH and the temperature to allow complete reaction.
  • the preparation steps can be reasonably increased or simplified according to actual needs, to obtain a zinc peptide product having the same effect as the above-mentioned zinc ion complex.
  • the pH is 5.0 to 6.5
  • the reaction temperature is 60° C. to 90° C.
  • the above-described zinc ion solution is selected from the group consisting of zinc chloride solution, zinc sulfate solution, zinc oxide solution or zinc acetate solution; and the concentration in the solution is 0.1 to 1.0 mmol/L.
  • a fourth aspect of the present invention provides:
  • the present invention provides a peptide for complexing zinc ion and its complex, and the amino acid composition and the amino acid sequence of the peptide are identified, which promotes the development of its directed synthesis and preparation process, and further produce the peptide-zinc complex in bulk;
  • the peptide for complexing in the present invention is derived from soybean protein or peanut protein, which is an inherent component of food protein and has a super strong complexing effect with zinc ions.
  • FIG. 1 is a gel chromatography separation spectrogram of the peptide component SPIH2.
  • FIG. 2 is a sequence diagram of the amino acid composition of peptide for complexing F21 identified by HPLC (A) and LC-ESI/MS (B, C) methods.
  • FIG. 3 is a comparison chart of infrared spectrum FTIR of peptide for complexing zinc ion and peptide-zinc complex.
  • FIG. 4 is the first-order mass spectrum (A) and the second-order mass spectrum (B) for electrospray of the peptide-zinc complex.
  • FIG. 5 is H-NMR spectra of the peptide for complexing zinc ion (A) and the peptide-zinc complex (B).
  • This example provides a method for preparing a zinc ion complex, and the steps include: (1) Mixing soybean protein powder with pure water in a ratio of 1:50, adjusting the temperature to 50° C. and the pH value to 8.0, and adding ALcalase enzyme for enzymolysis;
  • step (2) Taking the enzymatic hydrolysate obtained in step (1) for inactivating the enzyme at 100° C. for 5 minutes, centrifuging, and taking the supernate to obtain a protein peptide solution;
  • step (3) Taking the protein peptide solution obtained in step (2) for ultrafiltering and fractionating, wherein the molecular weight cut offs of the ultrafiltration membrane are 5K Da, 1K Da, and 0.5K Da, respectively; and collecting the 1 to 5K Da peptide component SPIH2 with the strongest complexing rate of the zinc ion (see Table 1);
  • step (3) Taking the SPIH2 component obtained in step (3) for gel chromatography separation, screening and collecting the peptide component F2 with the strongest complexing ability with zinc ions (see FIG. 1 );
  • This example provides a method for preparing a zinc ion complex, and the steps include:
  • soybean meal for ultra-fine crushing or puffing followed by crushing to prepare soybean meal protein powder then mixing soybean protein powder with pure water in a ratio of 1:10, adjusting the temperature to 50° C. and pH value to 7.0, and adding Papain and Bacillus subtilis neutral protease in sequence for enzymolysis;
  • step (2) Taking the enzymatic hydrolysate obtained in step (1) for inactivating the enzyme at 100° C. for 5 minutes, centrifuging, and taking the supernate to obtain a protein peptide solution;
  • step (3) Taking the protein peptide solution obtained in step (2) for ultrafiltering and fractionating, wherein the molecular weight cut offs of the ultrafiltration membrane are 5K Da, 1K Da, and 0.5K Da, respectively; and collecting the 1 to 5K Da peptide component SPIH2 with the strongest complexing rate of the zinc ion (see Table 1);
  • step (3) Taking the SPIH2 component obtained in step (3) for gel chromatography separation, screening and collecting the peptide component F2 with the strongest complexing ability with zinc ions (see FIG. 1 );
  • This example provides a preparation method and use of a zinc ion complex, and the steps include:

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Abstract

The invention provides a peptide for complexing zinc ion, complex thereof and use therefor. The amino acid composition and sequence of the peptide for complexing zinc ion are Lys-Tyr-Lys-Arg-Gln-Arg-Trp (SEQ ID NO: 1). The peptide for complexing is derived from soybean or peanut, is an inherent component of foods, and has a super strong complexing effect with zinc ions.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • The present application is a Continuation Application of PCT Application No. PCT/CN2020/117329 filed on Sep. 24, 2020, which claims the benefit of Chinese Patent Application No. 202010073635.X filed on Jan. 22, 2020. All the above are hereby incorporated by reference in their entirety.
  • Reference to Sequence Listing
  • A sequence listing is submitted as an ASCII formatted text filed via EFS-Web, with a file name of “Sequence_listing.TXT”, a creation date of Jan. 27, 2022, and a size of 486 bytes. The sequence listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein.
    • TECHNICAL FIELD
  • The invention relates to the technical field of biochemical engineering, in particular to a peptide for complexing zinc ion, complex thereof and use therefor.
    • BACKGROUND
  • In China, the phenomenon of zinc deficiency is commonly seen among the crowd, and it is especially serious for children. Up to 39.6% of the children have different degrees of zinc deficiency, and the zinc deficiency rate is as high as 64.6% especially for children aged 3 to 4. For those aged 0 to 18, zinc deficiency ranks the first in trace element deficiencies with a zinc deficiency rate of 50% or more. Promoting the transport and absorption of the trace element zinc in the human body, and improving the absorption and the utilization rate of zinc are important scientific issues that have always been concerned with in the fields of food nutrition or functional foods. In addition, in order to avoid the crisis of illness caused by zinc deficiency in farmed livestock, poultry and aquatic animals, and so on, it is usually necessary to add zinc supplements to the feed. At present, inorganic zinc such as zinc oxide, zinc sulfate or zinc gluconate etc. is usually used as a zinc supplement for human or animal bodies.
  • Existing studies have found that it is organic zinc rather than inorganic zinc that actually plays a role inside the body. Organic zinc is closer to the functional form of zinc inside the organism, which can prevent the formation of insoluble substances inside the organism due to the supplement of inorganic zinc. Moreover, organic zinc has a higher biological efficiency than that of inorganic zinc, and only a trace amount of organic zinc is necessary to achieve the effect of zinc supplementation, which avoids the harm caused by excessive zinc inside the body due to poor absorption of inorganic zinc. Studies have shown that active peptides that have affinity with zinc ions (Zn2+), peptides for complexing zinc, and their complexes can both promote the transport and the absorption of zinc. Peptide-zinc complex belongs to the category of organic zinc, is a new type of trace element enhancer that promotes the body's zinc intake, and is more helpful to promote the transport and the absorption of zinc by the body's cells.
  • At present, there is a general lack of related products of peptides for complexing zinc and complexes thereof in the market. Safe and effective organic zinc supplement products are lacked especially in aspects of foods and animal feed. Therefore, providing a stable and effective peptide for complexing zinc ion and its complexes can make up for the gap in this field.
    • SUMMARY OF THE INVENTION
  • The purpose of the present invention is to provide a peptide for complexing zinc ion, the complex thereof and use therefor.
  • The technical solutions used in the present invention are:
  • A first aspect of the present invention provides:
  • a peptide for complexing zinc ion having an amino acid composition and sequence of SEQ ID NO:1: Lys-Tyr-Lys-Arg-Gln-Arg-Trp (KYKRQRW).
  • In some embodiments, the above-described peptide for complexing zinc ion is derived from soybean, peanut or chemical synthesis. Certainly, other alternative amino acid and protein synthesis methods in this field can be reasonably selected according to actual needs, to obtain the above-mentioned peptide for complexing zinc ion.
  • A second aspect of the present invention provides:
  • a method for preparing a peptide for complexing zinc ion comprises steps as follows:
  • (1) taking soybean or peanut materials, and adjusting temperature and pH to perform enzymolysis, to obtain a protein peptide solution; and
  • (2) taking the protein peptide solution obtained in step (1), and subjecting it to separating, screening, and purifying, to obtain a peptide component, wherein the enzyme used for the enzymolysis is one or more selected from the group consisting of ALcalase enzyme, papain and Bacillus subtilis neutral protease; and the peptide component contains the above-described peptide for complexing zinc ion.
  • Certainly, the preparation steps thereof can be reasonably increased or simplified according to actual needs, to obtain peptides with the same effect as the above-mentioned peptide component.
  • In some embodiments, the molecular weight of the peptide component in step (2) is 1 to 5 K Da; the content of Peptide is 15.58% to 17.18%; and the complexing rate of the zinc ion is 48.5% to 52.16%. Certainly, the molecular weight of the peptide component, the content of Peptide, and the complexing rate of the zinc ion can be reasonably selected according to actual needs.
  • In some embodiments, the way for the above-described separating, screening, and purifying in step (2) is one or more selected from the group consisting of ultrafiltration fractionation, gel chromatography and high performance liquid chromatography.
  • Certainly, other alternative means for separating, screening, and purifying in this field can also be reasonably selected according to actual needs.
  • In some embodiments, in step (1), the temperature is 35° C. to 55° C., and the pH is 6.0 to 8.5.
  • Certainly, the pH can be adjusted adaptively during selecting enzymes according to actual needs. Specifically, pH for the ALcalase enzyme is 7.0 to 8.5, pH for the papain is 6.5 to 7.5, and pH for the Bacillus subtilis neutral protease is 6.0 to 7.0.
  • Certainly, other alternative enzymes in this field can also be reasonably selected according to actual needs.
  • A third aspect of the present invention provides:
  • a zinc ion complex, the preparation method thereof is: taking the above-described peptide for complexing zinc ion or the above-described peptide component, adding a zinc ion solution, and adjusting the pH and the temperature to allow complete reaction.
  • Certainly, the preparation steps can be reasonably increased or simplified according to actual needs, to obtain a zinc peptide product having the same effect as the above-mentioned zinc ion complex.
  • In some embodiments, in the preparation reaction of the above-described zinc ion complex, the pH is 5.0 to 6.5, and the reaction temperature is 60° C. to 90° C.
  • Certainly, the pH and the temperature in the reaction can be adjusted reasonably according to actual needs.
  • In some embodiments, the above-described zinc ion solution is selected from the group consisting of zinc chloride solution, zinc sulfate solution, zinc oxide solution or zinc acetate solution; and the concentration in the solution is 0.1 to 1.0 mmol/L.
  • Certainly, other alternative solutions containing zinc ions with different concentrations can be reasonably selected according to actual needs.
  • A fourth aspect of the present invention provides:
  • uses of the above-described peptide for complexing zinc ion, the peptide component prepared by the above-described method, and the zinc ion complex in preparing foods and animal feed.
  • The beneficial effects of the present invention are:
  • 1. the present invention provides a peptide for complexing zinc ion and its complex, and the amino acid composition and the amino acid sequence of the peptide are identified, which promotes the development of its directed synthesis and preparation process, and further produce the peptide-zinc complex in bulk; and
  • 2. the peptide for complexing in the present invention is derived from soybean protein or peanut protein, which is an inherent component of food protein and has a super strong complexing effect with zinc ions.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a gel chromatography separation spectrogram of the peptide component SPIH2.
  • FIG. 2 is a sequence diagram of the amino acid composition of peptide for complexing F21 identified by HPLC (A) and LC-ESI/MS (B, C) methods.
  • FIG. 3 is a comparison chart of infrared spectrum FTIR of peptide for complexing zinc ion and peptide-zinc complex.
  • FIG. 4 is the first-order mass spectrum (A) and the second-order mass spectrum (B) for electrospray of the peptide-zinc complex.
  • FIG. 5 is H-NMR spectra of the peptide for complexing zinc ion (A) and the peptide-zinc complex (B).
    •  DETAILED DESCRIPTION
  • In order to make the purpose, technical solutions and technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, not for limiting the present invention.
  • The following examples all use the same peptide for complexing zinc ion, and its amino acid composition and amino acid sequence are Lys-Tyr-Lys-Arg-Gln-Arg-Trp (SEQ ID NO: 1).
  • TABLE 1
    Comparison of complexing rates of zinc of peptide segments with different molecular
    weights
    Protein Peptide Peptide Peptide Peptide
    peptide component component component component
    Items solution SPIH1 SPIH2 SPIH3 SPIH4
    Molecular / >5K 1 to 5K 0.5 to 1K <0.5K
    weight cut
    off (Da)
    Content of / 37.85 ± 1.98 16.38 ± 0.80 17.59 ± 0.55 28.16 ± 0.6
    Peptide ( % )
    complexing 30.96 ± 1.52c 36.95 ± 3.62b 50.33 ± 1.83a 36.84 ± 3.14b 10.31 ± 0.19d
    rate of zinc
    ( % )
  • Significant difference coefficient p<0.05.
    • Example 1
  • This example provides a method for preparing a zinc ion complex, and the steps include: (1) Mixing soybean protein powder with pure water in a ratio of 1:50, adjusting the temperature to 50° C. and the pH value to 8.0, and adding ALcalase enzyme for enzymolysis;
  • (2) Taking the enzymatic hydrolysate obtained in step (1) for inactivating the enzyme at 100° C. for 5 minutes, centrifuging, and taking the supernate to obtain a protein peptide solution;
  • (3) Taking the protein peptide solution obtained in step (2) for ultrafiltering and fractionating, wherein the molecular weight cut offs of the ultrafiltration membrane are 5K Da, 1K Da, and 0.5K Da, respectively; and collecting the 1 to 5K Da peptide component SPIH2 with the strongest complexing rate of the zinc ion (see Table 1);
  • (4) Taking the SPIH2 component obtained in step (3) for gel chromatography separation, screening and collecting the peptide component F2 with the strongest complexing ability with zinc ions (see FIG. 1);
  • (5) Taking the peptide component F2 for separation and purification by high performance liquid chromatography (HPLC), to obtain the single peptide component for complexing F21 with the strongest complexing ability with zinc ion, and then using LC-ESI/MS for amino acid sequence analysis to identify the amino acid sequence of the peptide for complexing to be Lys-Tyr-Lys-Arg-Gln-Arg-Trp (i.e. KYKRQRW (SEQ ID NO: 1)) (see FIG. 2); and
  • (6) Under conditions of a KYKRQRW (SEQ ID NO: 1) peptide concentration of 1 mg/mL, a zinc chloride ZnCl2 concentration of 0.5 mmol/L, a reaction temperature of 70° C., and pH=5.5, subjecting the peptide for complexing (KYKRQRW (SEQ ID NO: 1)) and zinc ions to complexing reaction so as to generate peptide-zinc complex (KYKRQRW (SEQ ID NO: 1)-Zn) (see FIG. 3, FIG. 4, and FIG. 5).
    • Example 2
  • This example provides a method for preparing a zinc ion complex, and the steps include:
  • (1) Taking soybean meal for ultra-fine crushing or puffing followed by crushing to prepare soybean meal protein powder, then mixing soybean protein powder with pure water in a ratio of 1:10, adjusting the temperature to 50° C. and pH value to 7.0, and adding Papain and Bacillus subtilis neutral protease in sequence for enzymolysis;
  • (2) Taking the enzymatic hydrolysate obtained in step (1) for inactivating the enzyme at 100° C. for 5 minutes, centrifuging, and taking the supernate to obtain a protein peptide solution;
  • (3) Taking the protein peptide solution obtained in step (2) for ultrafiltering and fractionating, wherein the molecular weight cut offs of the ultrafiltration membrane are 5K Da, 1K Da, and 0.5K Da, respectively; and collecting the 1 to 5K Da peptide component SPIH2 with the strongest complexing rate of the zinc ion (see Table 1);
  • (4) Taking the SPIH2 component obtained in step (3) for gel chromatography separation, screening and collecting the peptide component F2 with the strongest complexing ability with zinc ions (see FIG. 1);
  • (5) Subjecting the peptide component F2 to separation and purification by high performance liquid chromatography (HPLC), to obtain the single peptide component for complexing F21 with the strongest complexing ability with zinc ion, and then using LC-ESI/MS for amino acid sequence analysis, to identify the amino acid sequence of the peptide for complexing to be Lys-Tyr-Lys-Arg-Gln-Arg-Trp (i.e. KYKRQRW (SEQ ID NO: 1)) (see FIG. 2); and
  • (6) Under conditions of a KYKRQRW (SEQ ID NO: 1) peptide concentration of 1 mg/mL, a zinc chloride ZnCl2 concentration of 0.5 mmol/L, a reaction temperature of 70° C., and pH=5.5, subjecting the peptide for complexing (KYKRQRW (SEQ ID NO: 1)) and zinc ions to complexing reaction so as to generate peptide-zinc complex (KYKRQRW (SEQ ID NO: 1)-Zn) (see FIG. 3, FIG. 4, and FIG. 5).
    • Example 3
  • This example provides a preparation method and use of a zinc ion complex, and the steps include:
  • (1) According to the amino acid composition and sequence of the peptide for complexing zinc ion, using a solid phase peptide synthesis method to prepare a peptide for complexing zinc ion (Lys-Tyr-Lys-Arg-Gln-Arg-Trp) with a purity of 95% or more (see FIG. 2); and
  • (2) Preparing a peptide for complexing zinc ion (100 μM), dissolving it in a 50 mM phosphate buffer solution (pH 7.2) containing ZnCl2 (250 μM), and stirring the mixture at room temperature to equilibrate for 1 hour; using a semi-permeable membrane with a molecular weight cut-off of 500 Da for dialysis for 5 hours to remove unbound zinc, collecting the retentate (complex), and freeze-drying to prepare the peptide-zinc complex powder.
  • The above-mentioned examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned examples, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should all be equivalent replacement modes, and they are all included in the protection scope of the present invention.

Claims (15)

1. A peptide for complexing zinc ion, characterized in that, the peptide for complexing zinc ion has an amino acid sequence of SEQ ID NO: 1.
2. The peptide for complexing zinc ion according to claim 1, characterized in that, the peptide for complexing zinc ion is derived from soybean, peanut or chemical synthesis.
3. A method for preparing a peptide for complexing zinc ion, comprising the following steps:
(1) taking soybean or peanut materials, and adjusting temperature and pH to perform enzymolysis, to obtain a protein peptide solution; and
(2) taking the protein peptide solution obtained in step (1), and subjecting it to separating, screening, and purifying, to obtain a peptide component,
wherein the enzyme used for the enzymolysis is one or more selected from the group consisting of ALcalase enzyme, papain and Bacillus subtilis neutral protease; and
the peptide component contains the peptide for complexing zinc ion according to claim 1.
4. The method for preparing according to claim 3, characterized in that, the molecular weight of the peptide component obtained in step (2) is 1 to 5 K Da; the content of Peptide is 15.58% to 17.18%; and the complexing rate of the zinc ion is 48.5% to 52.16%.
5. The method for preparing according to claim 3, characterized in that, the way for separating, screening, and purifying in step (2) is one or more selected from the group consisting of ultrafiltration fractionation, gel chromatography and high performance liquid chromatography.
6. The method for preparing according to claim 3, characterized in that, in step (1), the temperature is 35° C. to 55° C., and the pH is 6.0 to 8.5.
7. A zinc ion complex, characterized in that, the zinc ion complex is prepared by the following method: taking the peptide for complexing zinc ion according to claim 1, adding a zinc ion solution, and adjusting the pH and the temperature to allow complete reaction.
8. The zinc ion complex according to claim 7, characterized in that, the pH is 5.0 to 6.5, and the reaction temperature is 60° C. to 90° C.
9. The zinc ion complex according to claim 7, characterized in that, the zinc ion solution is selected from the group consisting of zinc chloride solution, zinc sulfate solution, zinc oxide solution or zinc acetate solution; and the concentration in the solution is 0.1 to 1.0 mmol/L.
10. A zinc ion complex, characterized in that, the zinc ion complex is prepared by the following method: taking the peptide for complexing zinc ion prepared by the method according to claim 3, adding a zinc ion solution, and adjusting the pH and the temperature to allow complete reaction.
11. The zinc ion complex according to claim 10, characterized in that, the pH is 5.0 to 6.5, and the reaction temperature is 60° C. to 90° C.
12. The zinc ion complex according to claim 10, characterized in that, the zinc ion solution is selected from the group consisting of zinc chloride solution, zinc sulfate solution, zinc oxide solution or zinc acetate solution; and the concentration in the solution is 0.1 to 1.0 mmol/L.
13. Use of the peptide for complexing zinc ion according to claim 1 in preparing foods and animal feed.
14. Use of the peptide component prepared by the method according to claim 3, in preparing foods and animal feed.
15. Use of the zinc ion complex according to claim 7 in preparing foods and animal feed.
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